Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

Abstract

Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD ( 80 ), RpoN ( 54 ), and FliA ( 28 ), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaB and flgE) and FliA-dependent (flaA) genes. The MS ring (encoded by fliF) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ϳ4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components (fliM and fliY) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA. Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins (fliE and flgBC) were deleted, while transcript levels of flaA was reduced ϳ2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure. T he bacterial flagellum is a complex nanomachine powered by an ion-driven rotary motor consisting of about 30 different types of proteins whose copy numbers range from a few to thousands (Fig. 1). The flagellum consists of three basic structures referred to as the basal body, hook, and filament (1). The basal body is an intricate complex that consists of the flagellar rod, rings, a motor, a switch complex, and a specialized type III secretion system (T3SS) that transports flagellar proteins across the cell membrane (1-3). The T3SS, also referred to as the flagellar protein export apparatus, consists of integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) which form an export pore located within the inner membrane, as well as cytoplasmic components (FliI, FliH, and FliJ) that deliver protein substrates to the export pore (4, 5). During flagellar assembly, the export apparatus initially transports rod-and hook-type substrates across the cell membrane into the lumen of the nascent flagellum (6, 7). Upon completion of the mature hook-basal body (HBB) structure, the export apparatus undergoes a conformational change that is accompanied by a switch in substrate specificity to filament-type substrates.Biogenesis of the bacterial flagellum involves a transcriptional hierarchy that is responsive to checkpoints in assembly so that expression of specific flagellar genes occurs as t...

A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates

“…Additionally, we found that V. cholerae FliF mutants that likely fail to form an MS ring were defective in FlrBC TCS activity. These findings are similar to our previous studies in C. jejuni and what others have reported in H. pylori (32,40,41,43). Furthermore, we accumulated evidence that for at least P. aeruginosa (and possibly V. cholerae), more domains of the FliG rotor were required for flagellum-associated TCS activity than in C. jejuni.…”

“…Detection of this regulatory checkpoint by an orthologous FlgSR TCS also may occur in Helicobacter pylori, a lophotrichous epsilonproteobacterium closely related to C. jejuni, for transcription of 54 -dependent rod and hook genes (34)(35)(36)(37). Genetic analyses indicate that H. pylori FlgSR TCS activity for rod and hook gene transcription is also dependent on fT3SS, MS ring, and C ring proteins, suggesting that this TCS in H. pylori also senses the formation of a competent fT3SS (38)(39)(40)(41)(42)(43).…”

Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

“…H. pylori also appears to have different requirements of C-ring proteins for flagellar assembly from those to C. jejuni. H. pylori fliM and fliY mutants are severely defective for flagellar biogenesis, but a fliN mutant is much less so; all of these H. pylori mutants are generally less flagellated than are the respective C. jejuni mutants (56,57,88). Our work combined with this analysis in H. pylori, a closely related epsilonproteobacterium, demonstrate that flagellar motors are composed of different components across bacterial species, which result in motors with different assemblies of substructures, alteration of motor output for propulsion, and roles outside flagellar motility.…”

Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis

“…The flagellum is structurally composed of the basal body, hook, and filament (Ran Kim and Haeng Rhee, 2003; Hirano et al, 2009; Evans et al, 2014; Gao et al, 2014; Tsang and Hoover, 2015). The basal body serves as a rotation axis and consists of the MS-ring, C-ring, PL-ring, and rod.…”